1. Fish Pituitary gland

2. Structure: Pituitary gland is an endocrine gland situated on the ventral side of the brain It is a small, soft, whitish body whose size and shape vary with species It is more or less round in carps; oval in catla and rohu and pear-shaped in mrigal The pituitary is located in a concave cavity known as Sella turcica and enclosed by by a thin membrane known as durameter It may be attached to the brain by a short stalk called the Infundibular stalk.

4. Types of pituitary glands Based on the presence or absence of the stalk, the pituitary is classified into Leptobasic (with stalk) and Platybasic (without stalk) pituitary Leptobasic pituitary – eg. Carps and catfishes Platybasic pituitary – eg. Glassfish (Ambasis spp.) The teleost pituitary comprises of two parts – the glandular part (the adenohypophysis) and the nervous part (the neurohypophysis)

5. Collection of pituitary gland: Fish pituitary gland can be collected by dissecting and removing a portion of the scalp or through the Foramen magnum (1)Dissecting and removing a portion of the scalp : In this method, the brain case (cranium) is obliquely cut using a butcher’s knife/hand saw/bone cutter and the scalp removed The brain is then exposed by removing grey matter and fatty substance with foreceps and cotton Figure: Making an oblique cut in the cranium Figure: Fatty tissue and grey matter exposed

6. The anterior end (optic and olfactory nerves) of the brain is cut and the entire brain is lifted up and laid back, thus exposing the pituitary under a membrane After removing the membrane and the fluid, the pituitary is lifted up by inserting the blunt end of the foreceps and carefully transferred to a vial containing a preservative Figure: The brain being exposed Figure: The pituitary seen as a small whitish body Figure: The pituitary mounted on to a hand

7. (2) Through the Foramen magnum: Foramen magnum is a large posterior aperture of the skull through which the spinal cord passes The grey matter and fatty substance are first removed with the help of foreceps and cotton (they are pulled out posteriorly) The brain is then exposed The anterior end (optic and olfactory nerves) of the brain is cut and the entire brain is lifted up and laid back, thus exposing the pituitary

8. After removing the fluid the membrane, the pituitary is lifted up by inserting the blunt end of the foreceps and carefully transferred to a vial containing a preservative The first method is commonly practiced even though the second method is less time consuming and a large number of glands can be collected within a short time, with a good resale value of the fish Even an untrained person can practise the first method

9. Preservation of pituitary gland: 1.Preservation in absolute alcohol : 2.The gland, after collection, is immediately transferred to a vial/phial containing fresh absolute alcohol (ethanol) After 24 hours, the alcohol is removed and fresh alcohol is added and stored at room temperature or in a refrigerator 2. Preservation in acetone: Immediately after collection, the pituitary gland is kept in ice-chilled acetone and stored in a refrigerator for 2-3 days After this period, the acetone is changed and the gland stored in a refrigerator Both absolute alcohol and acetone have defattening and dehydrating effect 3. Immediate freezing The collected glands are frozen immediately and stored in a freezer

10. Preparation of fish pituitary extract for injection The extract preparation should be carried out just before injection The required quantity of glands is taken out of vial and they are dried on a filter paper by allowing the alcohol to evaporate The glands are then homogenized with distilled water or saline in a tissue homogenizer If acetone-dried glands are used, they can directly be taken for maceration One-third of the media is used for homogenization, while the remaining two- third is used for rinsing the homogenizer and the glass rod Recommended dilution rate is 20-30 mg in 1 ml of the media The extract is centrifuged at 5,000 rpm for 5 minutes The clear supernatant solution containing gonadotropins is taken in syringe for injection

11. Dosage of pituitary extract Assessment of proper dosage is most important for successful spawning Female – 2 injections, male- 1 injection, i.e. at the time of second injection to the female I Dose or Provocative or preliminary dosage and II Dose or effective or resolving dosage The interval between the two doses is 6 hours. Carp glands to major carps: FemaleMale I Dose2-3 mg/kg b.w.nil II Dose5-8 mg/kg b.w.2-3 mg/kg b.w. Carp glands to exotic carps: FemaleMale I Dose4-6 mg/kg b.w.nil II Dose10-16 mg/kg b.w.4-6 mg/kg b.w. Catfish glands to major carps: FemaleMale I Dose10 mg/kg b.w.nil II Dose20 mg/kg b.w.10 mg/kg b.w. Catfish glands to exotic carps: FemaleMale I Dose20 mg/kg b.w.nil II Dose40 mg/kg b.w.20 mg/kg b.w.

12. Types of injection: Homoplastic injection : Injecting pituitary from one fish to another fish closely related to the donor fish. E.g. carp pituitary gland extract to carps Heteroplastic injection : Injecting pituitary from one fish to another fish distantly related to the donor fish. E.g. carp pituitary gland extract to catfish and vice versa